Anti‐Toxoplasma gondii activity of rose hip oil–solid lipid nanoparticles

Abstract Toxoplasma gondii is a highly prevalent pathogen, reported from almost all geographical regions of the world. Current anti‐T. gondii drugs are not effective enough in immunocompromised patients, encephalitis, chorioretinitis, and congenital toxoplasmosis. Therefore, the prescription of these drugs has been limited. Rose hip oil (RhO) is a natural plant compound, which shows antibacterial, anticancer, and anti‐inflammatory activities. In the current study, the anti‐T. gondii and cell toxicity effects of solid lipid nanoparticles (SLNs) loaded by RhO (RhO‐SLNs) were evaluated. Emulsification sonicated‐homogenization method was used to prepare SLNs. RhO‐SLNs were characterized, and their anti‐T. gondii and cell toxicity effects were evaluated using in vitro analyses. The particle size and the zeta potential of the nanoparticles were 152.09 nm and −15.3 mV nm, respectively. The entrapment efficiency percentage was 79.1%. In the present study, the inhibitory concentration (IC)50 against T. gondii was >1 μg/mL (p‐value <.0001). The cell toxicity assay showed cytotoxicity concentration (CC)50 >10 mg/mL (p‐value = .017). In addition, at least 75% of T. gondii‐infected Vero cells remained alive at concentrations >10 mg/mL. The concentration of 1 mg/mL showed highest anti‐Toxoplasma activity and lowest cell toxicity against the Vero cell. Our findings suggest that carrying natural plant compounds with SLNs could be considered an effective option for treatment strategies against T. gondii infections.

Medicinal plants are the primary source of medicine, which have traditionally been employed for the treatment of human diseases for thousand years, all over the world.These natural products have been proved to be an alternative for chemical agents in the pharmaceutical industry (Molaei et al., 2021;Petrovska, 2012;Sofowora et al., 2013).Recently, natural extracts of plants have been considered as anti-Toxoplasma components (Choi et al., 2013;Kavitha et al., 2012;Khamesipour et al., 2022;Mohammad Rahimi et al., 2020;Nemati et al., 2022).Rosa rubiginosa (Rosa eglanteria L.), known as rose hips (Rosa spp., Rosaceae), are wild rose bushes with more than 100 species, which are widely used in the preparation of cosmetic products and the food industry (Franco, Pinelo, et al., 2007;Loetscher et al., 2013;Santana et al., 2016).Rose hip fruits have been very popular in different countries due to their antioxidant properties and having various minerals, high amount of vitamin C, ascorbic acid, carotenoids, and phenols (Demir et al., 2014;Ercisli, 2007;Franco, Pinelo, et al., 2007;Franco, Sineiro, et al., 2007;Roman et al., 2013).Many in vivo and in vitro studies have been conducted on the different compounds of rose hip oil (RhO) extract (Mármol et al., 2017;Yi et al., 2007).A significant effect of RhO extract was described in the treatment of lesions such as eczema and trophic skin injuries (Lei et al., 2019).Moreover, antibacterial (Rovnã et al., 2015), anticancer (Tumbas et al., 2012), antioxidant (Abdelbaky et al., 2021), and anti-inflammatory (Yan et al., 2013) properties of rose hip oil extracts have been evaluated.However, there is no evidence indicating antiparasitic effects of rose hip extracts.
In recent years, the development of nanotechnology sciences has resulted in the designing of novel drug delivery carriers, leading to development of pharmaceutical and cosmetic-sanitary products (Cai & Chen, 2007;Zhang et al., 2008).Liposomes, microemulsions, multiple emulsions, and solid particles are the most popular drug delivery carriers.In the early 1990s, solid lipid nanoparticles (SLNs), as a colloidal carrier with a particle size of 50-1000 nm, were designed and manufactured (Müller et al., 2002).SLNs are prepared from physiological lipids and dispersed well in aqueous surfactant solution (Musielak et al., 2022).The advantages of SLNs are small size, high surface-to-volume ratio, phase interaction at the particle level, high drug carrying capacity, appropriate release profile, ability to be used in the field of targeted drug delivery, high physical stability, and high biocompatibility (Ezzati Nazhad Dolatabadi et al., 2015;Uner & Yener, 2007).In addition, SLNs reduce the toxicity and degradation of a therapeutic agent, while increasing the clearance rate of the drug (Ghasemiyeh & Mohammadi-Samani, 2018).
Although antibacterial effects of RhO have been evaluated, there is no evidence indicating the application of this extract on parasites like Toxoplasma.In addition, employing lipid nanocarriers like SLNs can increase efficiency and decrease cell toxicity of herbal extracts.Therefore, employing a new formulation based on SLNs for target delivery of RhO seems to be novel in the drug delivery area.
In this study, we evaluated the effect of RhO-SLNs against T. gondii RH strain and Vero cell line.

| Particle size, polydispersity index (PDI) and zeta potential
A Zetasizer 1,033,439 (Malvern Instrument, UK) was utilized to determine the particle size and zeta potential of nanoparticles.In this regard, 20 μL of the sample was suspended in 1 mL of doubledistilled water and the average particle size was calculated as mentioned previously (Nemattalab et al., 2022).

| Transmission electron microscopy (TEM) and entrapment efficiency (EE)
Upon uranyl acetate staining and former resin coating, the shape and morphology of nanoparticles were characterized using a TEM microscope (Zeiss-EM10C-100 KV, Germany).To calculate the EE percentage, 1 g of RhO-SLN was dispersed in 10 mL of distilled water (containing 2% v/v tween 80), centrifuged at 6000 rpm for 20 min at 25°C, and the not encapsulated RhO and the entrapped oil percentage was calculated as mentioned previously (Nemattalab et al., 2022;Pinheiro et al., 2020).

| Release kinetics
To evaluate the release kinetics of the drug, 1.5 g of RhO-SLNs was packed into a dialysis bag with a molecular weight cutoff of 14 kDa (Sigma, Steinheim, Germany).The bag was immersed in receptor medium (100-mL distilled water containing 2% v/v tween 80), and the analyses of the RhO content were performed at different time points (Nemattalab et al., 2022;Wakade & Shende, 2021), based on available mathematical kinetic models (Mortazavi & Mortazavi, 2020).
The cell viability percentage was calculated as mentioned elsewhere (Nemati et al., 2022).
Tachyzoites without any treatment were considered a negative control.The intracellular anti-Toxoplasma activity of RhO-SLNs was evaluated (Nemati et al., 2022).Briefly, 1 × 10 4 of Vero cells were seeded in a 96-well plate containing DMEM, supplemented with 10% FBS, without antibiotics.After 24-h incubation at 37°C and 5% CO 2 , 1 × 10 4 of tachyzoites of T. gondii RH strain (MOI = 1) were added to the confluent Vero cells and incubated for an additional 24 h at 37°C and 5% CO 2 .The six concentrations of the RhO-SLNs were added to the T. gondii-infected Vero cells, and after incubation for an additional 24 h, absorbance intensity was read at wavelength 570 nm using ELISA reader (LX800; Biotec, Winooski, VA, USA).All experiences were done in duplicate.The anti-Toxoplasma activity to the cell viability proportion was calculated to indicate the best concentration of the RhO-SLNs with highest anti-Toxoplasma activity and lowest cell toxicity (Mohammad Rahimi et al., 2020;Nemati et al., 2022).
GraphPad Prism software (version 8.3.0.538) was used for statistical analysis and one-sample t-test was employed to evaluate the statistical correlations among samples.

TA B L E 1
Release kinetic parameters for RhO-SLNs based on different mathematical models.

Formulation Zero order
First order Higuchi Korsmeyer-Peppas Hixson-Crowell

| RhO-SLN preparation and characterization
The average particle size and the zeta potential of RhO-SLNs were 152.09nm and − 15.3 mV, respectively (Figure 1a).The relatively high surface charge leads to lower particle aggregation and better size stability due to stronger electrostatic repulsion between particles.
The TEM analysis of RhO-SLNs presented smooth nanoparticles with clear edge (Figure 1b).The entrapment efficiency was 79.1%, indicating lipophilic properties of the RhO-SLNs.

| Release kinetics
The formulation of RhO released from SLNs was performed.Maximum cumulative release reached 97% in 48 h, which reveals a relatively controlled release of RhO over time.The initial burst release at 6 h was 53.71% (Figure 2).The first-order model showed a regression coefficient with R 2 = 0.9909 (Table 1), indicating the effect of primary concentration of RhO on the release rate, which means the release rate can be modulated with primary RhO concentrations.

| Cell toxicity of the RhO-SLNs
The cytotoxicity concentration (CC) 50 for the RhO-SLNs was at the concentration >10 mg/mL.Accordingly, at least 60% of Vero cells remained viable in a concentration of more than 10 mg/mL.

| Anti-intracellular Toxoplasma activity of RhO-SLNs
The results showed that less than 20% of Toxoplasma-infected cells were dead in concentrations >1 μg/mL (Table 2).The viability of T.

| Anti-Toxoplasma activity of RhO-SLNs and ratio analysis
The tachyzoites were checked by a microscopic examination.
Accordingly, alive tachyzoites were oval [crescent] shaped with smooth surface, while dead tachyzoites were wrinkled.The RhO-SLNs showed an anti-Toxoplasma activity.Accordingly, at least 80% of T. gondii were dead in all concentrations with an inhibitory concentration (IC) 50 >1 μg/mL.
The high and low inhibitory effects of RhO-SLNs were 93.050 ± 9.83% and 81.200 ± 2.97% at 1 mg/mL and 1 μg/mL, respectively.In addition, statistical evaluations resulted in a significant concentration-dependent anti-Toxoplasma activity of the RhO-SLNs (p-value <.0001) (Figure 3b, Table 3).The ratio analysis suggested that the concentration of 1 mg/mL with a ratio value of 0.97 had the TA B L E 2 Anti-Toxoplasma activity of different concentrations of RhO-SLNs.

| DISCUSS ION
In past years, herbal medicine, based on the natural plant compounds, has been developed as an alternative remedy with lower side effects than chemical agents, for a broad spectrum of communicable and noncommunicable diseases (Firenzuoli & Gori, 2007;Welz et al., 2018).Numerous studies have evaluated the effectiveness of herbal extracts or their components on infectious diseases (Chaughule & Barve, 2023;Pinn, 2001).Toxoplasmosis is a highly prevalent infectious disease, which could be fatal in subjects with immune dysfunctions such as HIV/AIDS patients and organ transplant recipients (Wang et al., 2017).Toxoplasmosis in immunedeficient patients is associated with reactivation of latent infection, which leads to a wide variety of manifestations (Agrawal et al., 2014;Da Cunha et al., 1994).Therefore, immunocompromised patients are recommended to be treated against toxoplasmosis.Due to the side effects, resistance to current drugs, and limited efficacy, clinical treatment of toxoplasmosis and developing reliable and affordable drugs are still considerable concerns (Aspinall et al., 2002;Dunay et al., 2018;Meneceur et al., 2008).Recently, natural plant extracts and components have extremely been experienced in toxoplasmosis, which is mostly suggested as an alternative remedy with high activity, high efficiency, and low toxicity.Up to now, components and/or extracts of Artemisia annua (Secrieru et al., 2020), Myristica fragrans and Zingiber officinale Roscoe (Choi et al., 2013), Dracocephalum kotschyi (Khamesipour et al., 2021), Azadirachta indica (Nemati et al., 2022), Cinnamomum camphora (Alanazi & Almohammed, 2022), Lippia multiflora and Combretum micranthum (Benoit-Vical et al., 2000), Eurycoma longifolia (Kavitha et al., 2012), Sorghum bicolor (Abugri et al., 2016), and Mentha pulegium L. and Rubus idaeus L (Mohammad Rahimi et al., 2020) have been reported to be effective on toxoplasmosis.
A number of studies have investigated the anti-Toxoplasma activity of plant extracts and products, which have presented the ability of these natural compounds to inhibit the proliferation of Toxoplasma in vitro and in vivo (Khamesipour et al., 2022;Nemati et al., 2022;Rosenberg et al., 2019).However, the effects of the rose hip extract on T. gondii are not clear.Many nanotechnology-based formulations have been reported to improve the safety and efficacy of drugs in treating toxoplasmosis in vitro and in vivo (Assolini et al., 2017;Leyke et al., 2012).Teimouri et al. (2018) investigated the potential of various molecular weights and concentrations of chitosan nanoparticles (CS NPs), against tachyzoites of T. gondii RH strain.Their results showed that CS NPs were properly formulated and can be used as an alternative natural medicine in the treatment of toxoplasmosis.
During the last decades, nanotechnology has been widely developed in drug delivery systems.Nanoparticles with high surfaceto-volume ratio provide higher surface area and dissolution rate for particles (Barbosa et al., 2019).In addition, employing suitable nanocarriers overcomes the solubility and bioavailability limitations of conventional drugs (Alshawwa et al., 2022;Arshad et al., 2021;Din et al., 2017).
Nanocarriers increase the solubility of the hydrophobic agents and im- Nevertheless, encapsulation of plant extracts in SLNs, as a robust nanocarrier, has already been employed to overcome the limitations due to the multidrug resistance and to improve target delivery of antibiotics (Christaki et al., 2020;González-Paredes et al., 2019).Few studies have combined herbal extracts with SLNs for the treatment of parasites.Nemati et al. (2022) fabricated neem oil-SLNs (NeO-SLNs) and showed high ability of the nanodrug in killing of T. gondii tachyzoites in a concentration of 100 μg/mL with cell toxicity lower than 20%.According to our findings, RhO-SLNs in a concentration of 1 mg/mL showed higher anti-Toxoplasma activity with cell toxicity lower than 10%.Comparing the findings of ratio analysis with our previously published study suggests that the best concentration for RhO-SLNs was 1 mg/mL, while for NeO-SLNs, it was 100 μg/ mL.This finding suggests that although the anti-T.gondii effects are lower, the cell biosafety of RhO-SLNs is higher than NeO-SLNs.In addition, the EE% of RhO-SLNs was 79.1%, while it was 71.6% for NeO-SLNs (Nemati et al., 2022).

| CON CLUS ION
Our findings suggest the therapeutic aspects of RhO-SLNs.RhO-SLNs exhibited potent anti-Toxoplasma activity while its cell toxicity on Vero cells was acceptable.This study is a primary experiment, which suggested that encapsulation by a nano-vehicle such as SLNs may increase antimicrobial activity and decrease cell toxicity of herbal extracts.
RhO-SLNs were added to each experimental tube containing 2 × 10 4 of fresh tachyzoites.After incubation at 37°C for 2 h, the viability F I G U R E 1 RhO-SLN characterization based on (a) particle size analysis by DLS and (b) TEM two-dimensional morphological description.F I G U R E 2 Cumulative release profile of RhO from SLNs during 48 h.

F
I G U R E 3 Biological assays of the RhO-SLNs on (a) Vero cell line, which shows a statistically significant decrease in the toxicity of RhO-SLNs, regarding the concentration (p-value = .017),with a CC 50 >10 mg/mL and (b) statistically significant anti-Toxoplasma activity of RhO-SLNs (p-value <.0001) with IC 50 >1 μg/mL.(c) The concentration of 1 mg/mL of RhO-SLNs has high and low anti-Toxoplasma activity and cell toxicity, respectively.
prove target delivery of drugs(Jafarpour Azami et al., 2021).Polymeric nanoparticles, liposomes, dendrimers polymeric micelles, carbon nanotubes, and SLNs are well-known nanocarriers, of which SLNs are ideal candidates for developing enclosed carriers for different types of therapeutic agents(Bayón-Cordero et al., 2019).SLNs, as a proper option for medicinal purposes, present multiple advantages such as low toxicity, high stability, high biocompatibility and biodegradability, high release kinetics, and proper pharmacokinetics and pharmacodynamics(Nemati et al., 2024).In addition, SLNs are able to control the drug release(Scioli Montoto et al., 2020;Thorn et al., 2021).Due to their biopharmaceutical aspects, SLNs possess great advantages in certain specific therapeutic fields like cancer therapies (Scioli Montoto et al., 2020).da Rocha et al. (2020) investigated the antitumor effects of SLNs-docetaxel (SLN-DTX) to prevent tumor growth of 4 T1 murine mammary carcinoma cells.They showed that SLN-DTX not only exhibited high antitumor effects, but also reduced the tumor size compared to control, and prevented spontaneous lung metastasis test mice.From a toxicological point of view, Nassimi et al. (2009) showed high biosafety of SLNs for human lung as a desirable drug delivery system (DDS), based on the human alveolar epithelial cell line (A549) and murine precision-cut lung slices (PCLS).Furthermore, Khosravi et al. (2020) synthesized mannosylated paromomycin-loaded SLNs (PM-SLN-M) for evaluating anti-Toxoplasma effects.As a result, PM-SLN-M showed high anti-Toxoplasma activity and low cytotoxic effects on Vero cells compared to control groups.